Enhancing Graft-versus-Leukemia Responses by donor-derived CAR-modified CD8+ Memory Stem T cells

CAR T cell therapy has demonstrated curative potential in advanced B-cell malignancies; however, its efficacy is limited by insufficient in vivo expansion, persistence, and functional heterogeneity. Although numerous retrospective studies have correlated clinical response rates with the presence of early memory T cells, particularly memory stem T cells (TSCM), the frequency of these cells remains underrepresented in current approved CAR T products and their therapeutic relevance has not yet been prospectively evaluated. The primary aim of this thesis was to link CAR TSCM behavior to clinical response through in-depth longitudinal immunomonitoring of 11 patients treated with donor-derived CD8⁺ CAR TSCM cells, benchmarked against a cohort of 20 patients receiving conventional donor-derived CAR T cells. Mechanistic analyses showed that the higher complete remission rates at low doses and the improved safety profile of the TSCM-treated cohort were associated with enhanced CAR TSCM cell polyfunctionality, greater expansion, ability to generate both effector and self-renewing compartments through clonal succession, and a distinct cytokine profile characterized by IFNγ as the dominant inflammatory mediator versus IL-6 in the standard cohort. Importantly, treatment failure in the TSCM cohort was primarily driven by tumor- and host-mediated resistance mechanisms rather than intrinsic product defects. The secondary aim of the study was to investigate the biological mechanisms underlying the absence of graft-versus-host disease (GvHD) in both cohorts following donor-derived CAR T cell therapy. A cell-based platform was developed to expand rare, naturally occurring alloreactive T cells from the original alloHSCT donor and engineer them with a CD28-based CAR. We demonstrate that, while CAR stimulation alone was sufficient to induce severe alloreactive CAR T cell dysfunction, concomitant TCR engagement amplified activation, leading to rapid exhaustion and apoptosis. Modulation of CD28-mediated signaling (via AKT inhibition or a milder costimulatory domain such as 41BB) restored alloreactive CAR T cell functionality and persistence. Consistently, phenotypic and transcriptomic profiling along with longitudinal TCR tracking of post-infusion patient samples showed that dominant alloreactive CAR T clones displayed a dysfunctional signature at the peak of expansion, with only transient proliferation and limited persistence overtime. Collectively, by integrating longitudinal clinical immunomonitoring with mechanistic experimental modeling, this thesis demonstrates that TSCM enrichment in the infusion product improves CAR T cell efficacy and safety, and that CD28-based CAR signaling architecture constrains alloreactivity, ultimately limiting GvHD.